Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl(2). 2. These homogenates contained no detectable cytochrome c oxidase, succinate-cytochrome c oxidoreductase, succinate-ferricyanide oxidoreductase, l(+)-lactate-cytochrome c oxidoreductase or catalase. Cytochromes a+a(3) and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH- and NADPH-cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 10(5)g-min left 80-90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S(1)) together with 65 and 56% of the NADH- and NADPH-cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S(1) revealed the presence of a class of small particles containing NADPH-cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ;promitochondria' as described by other workers.     

The fractionation of nuclei from mammalian cells by zonal centrifugation

1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1.35 at 5 degrees .     

Subcellular fractionation of porcine neutrophils by nitrogen cavitation and sucrose-density-gradient centrifugation

Neutrophils, isolated in large quantities from porcine blood were disrupted by nitrogen cavitation and separated by differential centrifugation into a nuclear fraction and a post-nuclear supernatant. The latter was subfractionated by sucrose density gradient centrifugation into cytosol, a fraction consisting of membrane vesicles and two granule-rich fractions. The membrane fraction accounted for 1.9% of the protein in the post-nuclear supernatant, the light granule fraction for 2.2% and the dense granule fraction for 4.2%. Catalase, lactate dehydrogenase and malate dehydrogenase were largely confined to the cytosol. The dense granule fraction contained the highest quantities of the hydrolytic enzymes, although the membrane fraction was also rich in alkaline and acid phosphatase and gamma-glutamyl transpeptidase activities. Electron microscopy of the membrane fraction showed intact membrane vesicles, whereas the granular fractions consisted of electron-dense, membrane-bound granules. Two granular fractions were isolated which contained granules of differing size and density. 3H-labeled wheat germ agglutinin bound to the surface of intact neutrophils and when these were disrupted and fractionated the membrane fraction showed a specific binding activity 16-times greater than that of the cavitated sample. The membrane fraction interacted with the detergent digitonin and as a result underwent density perturbation increasing from 1.13 g X cm-3 to 1.18 g X cm-3. Dodecylsulphate-polyacrylamide gel electrophoresis showed the membrane fraction to consist of at least 40 protein bands, with relative molecular masses ranging from 200 000-16 000. The granule fractions contained less protein bands, with a protein composition quite distinct from that of the membrane fraction.     

Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.     

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-³H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.     

Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

Synchronization of Escherichia coli by zonal centrifugation.

A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.